The invention involves compositions and methods for treating diseases and the like by administering compounds that are both photosensitizers and sonosensitizers.
Treatment for cancer has traditionally encompassed three main strategies: surgery, chemotherapy, and radiotherapy. Although considerable progress in these areas has been attained, the search for more effective and safe alternative treatments continues. Lipson, et al. were the first to use photodynamic therapy (PDT), in 1966 at the Mayo Clinic [Proc. IX Internat. Cancer Congress, page 393 (1966)].
Since the advent of PDT, problems have been associated with photosensitizer use, including prolonged cutaneous phototsensitivity; the compositions are oligomeric mixtures of lipophilic molecules prone to molecular aggregation (with concomitant loss of photopotentiation); complicated pharmacokinetics; poor absorption and photoactivation in the xe2x80x9ctherapeutic windowxe2x80x9d (600 nm to 850 nm, i.e., visible red light). Furthermore, batch reproducibility, even in the clinical compositions, has been poor.
The photosensitizing properties of perylenequinonoid pigments (PQPs), such as hypocrellins, in biological systems have been recognized during the past two decades. See Diwu, et al., J. Photochem. Photobiol. A: Chem., 64:273 (1992); Zhang et al., (1989); and Wan, et al., xe2x80x9cHypocrellin A, a new drug for photochemotherapy,xe2x80x9d Kexue Tongbao (English edition) 26:1040 (1981).
Perylenequinones comprise a growing and highly diverse group of natural pigments, and they posses some unique chemical and biological properties. The natural perylenequinonoid pigments (PQP) identified to date include hypocrellins, cercosporin, phleichrome, cladochrome, elsinochromes, erythroaphins, and calphostins. Most of them are produced by a wide variety of molds. For their general chemical properties [see Weiss, et al., Prog. Chem. Org. Nat. Prod., 52:1 (1987) and Diwu, et al., Photochem and Photobiol., 52:609-616 (1990)]. PQP""s general photophysical and photochemical properties have been reviewed in Diwu, et al., Pharmac. Ther., 63:1 (1994). Hypocrellins belong to the general class of perylenequinonoid pigments, and include hypocrellin A (HA) and hypocrellin B (HB).
Because of the difficulty of collecting sufficient activated photosensitizer at the site of action, none of the previously known photosensitizers have gained widespread use as therapeutics.
The importance of sonodynamic therapy (SDT) lies ultimately in its similarity to PDT, an elegant and effective tumor treatment whose success is due to the use of light and drug in combination, i.e., two treatment elements, neither of which has toxic effects by itself (Marcus, 1992). PDT has mild side effects, destroys relatively little healthy tissue, and new photosensitizers with better therapeutic indices and improved clinical properties are being developed. The principal impetus for the development of SDT has been improvement upon PDT""s dosimetric shortcomings. PDT is currently restricted to use with superficial tumors. Its use on tumors deep within the body requires interstitial irradiation that increases the complexity of the treatment and compromises its noninvasive nature. SDT provides a means to reach such tumors, since ultrasound propagates easily through several centimeters of tissue, and like light, can be focused principally on the tumor mass where it activates the sonosensitizing compound. Targeted SDT offers the possibility of improving the tolerance of this therapy by further restricting its effects to the target tissue.
While these discoveries represent significant advances, two serious deficiencies remain in the development of experimental SDT. A substantial problem is the lack of sonodynamic agents with favorable clinical properties. Porphyrins are known to cause significant cutaneous photosensitivity (Estey et al., 1996), doxorubicin is cardiotoxic (Myers et al., 1976), and DMSO, DMF and MMF are hepatotoxic (Misik and Riesz, 1996). New sensitizers with better sonodynamic properties, which have milder side effects and which are rapidly cleared, would greatly improve the clinical application of SDT. A further problem is the lack of standardization in the conditions used for evaluating sonodynamic agents.
Potential sonodynamic agents have been tested following exposure to ultrasound intensities ranging from 0.25W/cm2 to 40W/cm2, and frequencies from 500 MHz to 1 MHz (Harrison et al., 1991; Sasaki et al., 1998). Though in vivo use would seem to require greater energies due to roughly isotropic dissipation of the ultrasonic energy, little effort has been made to compare experimental conditions in vitro with those in vivo. Where one group will find evidence of sonodynamic effect, different investigators do not under apparently similar conditions. Development of standard insonation and assay systems compatible with clinical use will permit a more rigorous assessment of the sonodynamic effects of current and future sonosensitizers.
Sonodynamic activation of sensitizers has been found to be useful since ultrasound has the appropriate tissue attenuation coefficient for penetrating intervening tissues to reach desired treatment volumes, while retaining the ability to focus energy on reasonably small volumes. Diagnostic ultrasound is a well accepted, non-invasive procedure widely used in the developed world, and is considered safe even for fetal imaging. The frequency range of diagnostic ultrasound lies between 100 kHz-12 MHz, while 50 kHz sound provides enough energy to effect cellular destruction through microregional cavitation.
Sonodynamic therapy provides treatment strategies unavailable in standard photodynamic therapy, due to the limited tissue penetration of visible light. One example would be the treatment of newly diagnosed breast cancer, where local and regional spread of micrometastatic disease remains clinically undetectable. Using immunoconjugates (anti-breast cancer Mabxe2x80x94sonosensitizer hybrids), it would be theoretically possible to selectively eradicate micrometastases in the absence of normal tissue damage.
Beyond these basic properties shared with other waves, ultrasound exhibits unique properties when propagating through water. Above a certain threshold intensity, propagation of ultrasound waves through water elicits an effect termed xe2x80x98cavitationxe2x80x99 (Rayleigh, 1917; Connolly and Fox, 1954). Cavitation involves the formation of small bubbles or xe2x80x98cavitiesxe2x80x99 in the water during the rarefaction half of the wave cycle, followed by the collapse of these bubbles during the compression half of the cycle (Putterman, 1995). Cavities focus the energy of the incident ultrasonic radiation by many orders of magnitude (Hiller et al., 1992). The consequence is that regions of cavitation in water are sites of extremely high temperature and pressure. Estimates of the temperatures generated in a collapsing cavity range from 5000K to 106K (Suslick et al. 1986; Flint and Suslick, 1991; Misik and Riesz, 1995; Kaiser, 1995).
The biological effects of exposure to ultrasound are the result of its physical and chemical effects. The most obvious biological effects of ultrasound treatment stem from heating of the medium through which it passes. Such heating is exploited during physiotherapy to help heal injured tissues. (Lehmann et al., 1967; Patrick, 1966), and has been investigated as a possible modality for tumor treatment. This is due to the sensitivity of many tumours to hyperthermia, a state in which tissue temperatures are elevated above 42xc2x0 C. (Doss and McCabe, 1976; Marmor et al., 1979; Sculier and Klastersky, 1981; Bleehen, 1982; Hynynen and Lulu, 1990). Ultrasound has also been used in combination with radiation therapy to improve treatment response in vivo compared to radiotherapy alone (Clarke et al., 1970; Repacholi et al., 1971; Mitsumori et al., 1996). A principal danger in the use of ultrasound for therapeutic purposes is the formation of xe2x80x98hotspotsxe2x80x99 due to regions of constructive interference and preferential absorption of ultrasonic energy by bone regions with low curvature radiixe2x80xa0 (Lehmann et al., 1967; Linke et al., 1973). These hotspots can cause serious damage to nearby tissues (Hill, 1968; Bruno et al., 1998).
As is the case of hematoporphyrin derivatives, natural PQPs do not themselves exhibit absorptivity longer than 600 nm, a characteristic that inherently predicts a decreased capability of activation as tissue depth increases beyond 3-5 mm. This means that the natural PQPs are not sufficiently strong for photodynamic therapy, and this limits their photodynamic therapy applications.
Deficiencies of current porphyrin and PQP photosensitizers for photodynamic therapy have stimulated the development of a series of second generation compounds which have improved properties with respect to light absorption in the red spectral range, purity, pharmacokinetics, and reduced cutaneous photosensitivity. These deficiencies also lead to investigating other forms of activating the sensitizer, e.g., activation using sound waves.
In accordance with the present invention, derivatives of perylenequinone pigments (PQPs) having both photosensitizing properties and sonosensitizing properties are used to treat diseases and other conditions. Moreover, the PQP derivatives of the present invention may be conjugated to a delivery moiety to enhance the ability of the PQP derivative to target pre determined cells or structures in vitro or in vivo.
The methods and compositions of the present invention, activated by light and/or sound, exhibit substantial absorption in the red spectral region or therapeutic frequencies of ultrasound; produce high singlet oxygen yield; can be produced in pure, monomeric form; may be derivatized to optimize properties of red light absorption, ultrasound activation, tissue biodistribution, and toxicity; have reduced residual cutaneous photosensitivity; and are rapidly excreted. They afford nuclear targeting by covalent attachment to DNA minor-groove binding agents, such as stapled lexotropins, to enhance phototoxicity. They are not genotoxic. This trait is important in the context of treatment-related secondary malignancies. Conjugation with transferrin affords specificity with respect to the treatment of a variety of diseases, including ovarian cancer and breast cancer. Conjugation with a bisphosphonate affords specificity with respect to the treatment of a variety of diseases, including any disease or condition that involves the bone matrix, e.g., bone metastases of breast and prostate cancer, or osteoporosis. Conjugation with a tumor binding peptide affords specificity with respect to the treatment of a variety of diseases, including those that involve specific cell surface carbohydrate antigens.
Many PQP properties are summarized in Diwu, et al., J. Photochem. Photobiol. A. Chem., 64:273 (1992). Some perylenequinones are also potent inhibitors of certain viruses, particularly human immunodeficiency virus (HIV), and also the enzyme protein kinase C (PKC). Both anti-HIV and anti-PKC activities of certain PQPs are light-dependent, a phenomenon implicated in the photodynamic therapy of cancers [Diwu, et al., Biochem. Pharmacol., 47:373-389 (1994)]. The Diwu et al paper also discloses the successful conjugation of HB to a protein.
The photosensitizing and sonosensitizing compounds of the present invention, when administered systemically, distribute throughout the body. Over a short period, ranging from hours to days, the compounds clear from normal tissues, but are selectively retained by rapidly proliferating cells (e.g., cancer cells or psoriasis lesions) for up to several days. The PQPs of the present invention are inactive and non-toxic until activated, e.g., exposed to light in a specific wavelength range or to sound in a specific frequency range.
The use of compounds that can be activated using two different activation protocols is therapeutically beneficial. Light, which can penetrate to a surface depth of about 5 mm to about 7 mm, can activate compounds for treating surface lesions or those target cells within a certain distance from a light source. Ultrasound, on the other hand, can penetrate deep within the body to treat deeply seated cells, such as tumor masses inaccessible to a source of light.
The compounds of the present invention are also beneficial therapeutically due to their dual selectivity. The compounds of the present invention are selective in their ability to preferentially localize the drug at the site of a predetermined target, such as a cancer cell, and they are selective in that precise delivery of light and/or sound can be confined to a specific area.
The methods and compositions of the present invention, when administered in vivo, such as intravenously, distribute throughout the body. In subsequent hours, and sometimes days, the compositions containing at least one perylenequinone derivative begin to clear from normal tissues, but are selectively retained for up to several days by hyperproliferating cells, such as cancer cells. The perylenequinone derivative remains inactive and non-toxic until it is activated. In accordance with the present invention, the perylenequinone derivative may be activated by light, by sound, or by light and sound. The hyperproliferating cells, now containing or contacted with a perylenequinone derivative, may be exposed to an activation source, e.g., light of an appropriate wavelength or sound of an appropriate frequency, or both. Exposing the site containing the hyperproliferating cells with the activation source permits selective activation of the retained perylenequinone derivative, which in turn initiates local necrosis or apoptosis in the hyperproliferating cell tissue leading to cell death.
In combination with the delivery system according to the present invention, the compositions and methods of the present invention permit increased selectivity by preferential localization of the perylenequinone derivative at the site of the targeted cells, and permit increased selectivity by confining the activation source to a specific area, e.g., light and/or sound confined to a discrete area.